Directional Guidance of Nicotiana alata Pollen Tubes in Vitro and on the Stigma1

Pollen tubes navigate the route from stigma to ovule with great accuracy, but the cues that guide them along this route are not known. We reproduced the environment on the stigma of Nicotiana alata by immersing pollen in stigma exudate or oil close to an interface with an aqueous medium. The growth of pollen in this culture system mimicked growth on stigmas: pollen grains hydrated and germinated, and pollen tubes grew toward the aqueous medium. The rate-limiting step in pollen germination was the movement of water through the surrounding exudate or oil. By elimination of other potential guidance cues, we conclude that the directional supply of water probably determined the axis of polarity of pollen tubes and resulted in growth toward the interface. We propose that a gradient of water in exudate is a guidance cue for pollen tubes on the stigma and that the composition of the exudate must be such that it is permeable enough for pollen hydration to occur but not so permeable that the supply of water becomes nondirectional. Pollen tube penetration of the stigma may be the most frequently occurring hydrotropic response of higher plants.

Arabidopsis Rho-Related GTPases: Differential Gene Expression in Pollen and Polar Localization in Fission Yeast1

The Rho small GTP-binding proteins are versatile, conserved molecular switches in eukaryotic signal transduction. Plants contain a unique subfamily of Rho-GTPases called Rop (Rho-related GTPases from plants). Our previous studies involving injection of antibodies indicated that the pea Rop GTPase Rop1Ps is critical for pollen tube growth. In this study we show that overexpression of an apparent Arabidopsis ortholog of Rop1Ps, Rop1At, induces isotropic cell growth in fission yeast (Schizosaccharomyces pombe) and that green fluorescence protein-tagged Rop1At displays polar localization to the site of growth in yeast. We found that Rop1At and two other Arabidopsis Rops, Rop3At and Rop5At, are all expressed in mature pollen. All three pollen Rops fall into the same subgroup as Rop1Ps and diverge from those Rops that are not expressed in mature pollen, suggesting a coupling of the structural conservation of Rop GTPases to their gene expression in pollen. However, pollen-specific transcript accumulation for Rop1At is much higher than that for Rop3At and Rop5At. Furthermore, Rop1At is specifically expressed in anthers, whereas Rop3At and Rop5At are also expressed in vegetative tissues. In transgenic plants containing the Rop1At promoter:GUS fusion gene, GUS is specifically expressed in mature pollen and pollen tubes. We propose that Rop1At may play a predominant role in the regulation of polarized cell growth in pollen, whereas its close relatives Rop3At and Rop5At may be functionally redundant to Rop1At in pollen.

Hexose Transport in Growing Petunia Pollen Tubes and Characterization of a Pollen-Specific, Putative Monosaccharide Transporter1

We investigated the molecular and physiological processes of sugar uptake and metabolism during pollen tube growth and plant fertilization. In vitro germination assays showed that petunia (Petunia hybrida) pollen can germinate and grow not only in medium containing sucrose (Suc) as a carbon source, but also in medium containing the monosaccharides glucose (Glc) or fructose (Fru). Furthermore, high-performance liquid chromatography analysis demonstrated a rapid and complete conversion of Suc into equimolar amounts of Glc and Fru when pollen was cultured in a medium containing 2% Suc. This indicates the presence of wall-bound invertase activity and uptake of sugars in the form of monosaccharides by the growing pollen tube. A cDNA designated pmt1 (petunia monosaccharide transporter 1), which is highly homologous to plant monosaccharide transporters, was isolated from petunia. Pmt1 belongs to a small gene family and is expressed specifically in the male gametophyte, but not in any other vegetative or floral tissues. Pmt1 is activated after the first pollen mitosis, and high levels of mRNA accumulate in mature and germinating pollen. A model describing the transport of sugars to the style, the conversion of Suc into Glc and Fru, and the active uptake by a monosaccharide transporter into the pollen tube is presented.

Pollen specific expression of maize genes encoding actin depolymerizing factor-like proteins.

In pollen development, a dramatic reorganization of the actin cytoskeleton takes place during the passage of the pollen grain into dormancy and on activation of pollen tube growth. A role for actin-binding proteins is implicated and we report here the identification of a small gene family in maize that encodes actin depolymerizing factor (ADF)-like proteins. The ADF group of proteins are believed to control actin polymerization and depolymerization in response to both intracellular and extracellular signals. Two of the maize genes ZmABP1 and ZmABP2 are expressed specifically in pollen and germinating pollen suggesting that the protein products may be involved in pollen actin reorganization. A third gene, ZmABP3, encodes a protein only 56% and 58% identical to ZmABP1 and ZmABP2, respectively, and its expression is suppressed in pollen and germinated pollen. The fundamental biochemical characteristics of the ZmABP proteins has been elucidated using bacterially expressed ZmABP3 protein. This has the ability to bind monomeric actin (G-actin) and filamentous actin (F-actin). Moreover, it decreases the viscosity of polymerized actin solutions consistent with an ability to depolymerize filaments. These biochemical characteristics, taken together with the sequence comparisons, support the inclusion of the ZmABP proteins in the ADF group.

Pex1, a pollen-specific gene with an extensin-like domain.

We report here the identification of a pollen-specific gene from Zea mays that contains multiple Ser-(Pro)n repeats, the motif found in the cell wall-associated extensins. Sequence analysis reveals that the encoded protein has a putative globular domain at the N terminus and an extensin-like domain at the C terminus. The Pex1 (pollen extensin-like) gene is expressed exclusively in pollen, not in vegetative or female tissues, and is not induced in leaves upon wounding. We propose that the encoded protein may have a role in reproduction, either as a structural element deposited in the pollen tube wall during its rapid growth or as a sexual recognition molecule that interacts with partner molecules in the pistil.

LeProT1, a Transporter for Proline, Glycine Betaine, and γ-Amino Butyric Acid in Tomato Pollen

During maturation, pollen undergoes a period of dehydration accompanied by the accumulation of compatible solutes.Solute import across the pollen plasma membrane, which occurs via proteinaceous transporters, is required to support pollen development and also for subsequent germination and pollen tube growth. Analysis of the free amino acid composition of various tissues in tomato revealed that the proline content in flowers was 60 times higher than in any other organ analyzed. Within the floral organs, proline was confined predominantly to pollen, where it represented >70 of total free amino acids. Uptake experiments demonstrated that mature as well as germinated pollen rapidly take up proline. To identify proline transporters in tomato pollen, we isolated genes homologous to Arabidopsis proline transporters. LeProT1 was specifically expressed both in mature and germinating pollen, as demonstrated by RNA in situ hybridization. Expression in a yeast mutant demonstrated that LeProT1 transports proline and γ-amino butyric acid with low affinity and glycine betaine with high affinity. Direct uptake and competition studies demonstrate that LeProT1 constitutes a general transporter for compatible solutes.

Expression-based and co-localization detection of arabinogalactan protein 6 and arabinogalactan protein 11 interactors in Arabidopsis pollen and pollen tubes

Arabinogalactan proteins (AGPs) are cell wall proteoglycans that have been shown to be important for pollen development. An Arabidopsis double null mutant for two pollen-specific AGPs (agp6 agp11) showed reduced pollen tube growth and compromised response to germination cues in vivo. A microarray experiment was performed on agp6 agp11 pollen tubes to search for genetic interactions in the context of pollen tube growth. A yeast two-hybrid experiment for AGP6 and AGP11 was also designed.

Reciprocal and advanced generation hybrids between Corymbia citriodora and C-torelliana: forestry breeding and the risk of gene flow

Corymbia F1 hybrids have high potential for plantation forestry; however, little is known of their reproductive biology and potential for genetic pollution of native Corymbia populations. This study aims to quantify the influence of reproductive isolating barriers on the success of novel reciprocal and advanced generation Corymbia hybrids. Two maternal taxa, Corymbia citriodora subsp. citriodora and Corymbia torelliana, were pollinated using five paternal taxa, C. citriodora subsp. citriodora, C. torelliana, one C. torelliana x C. citriodora subsp. citriodora hybrid and two C. torelliana x C. citriodora subsp. variegata hybrids. Pollen tube, embryo and seed development were assessed. Reciprocal hybridisation between C. citriodora subsp. citriodora and C. torelliana was successful. Advanced generation hybrids were also created when C. citriodora subsp. citriodora or C. torelliana females were backcrossed with F1 hybrid taxa. Prezygotic reproductive isolation was identified via reduced pollen tube numbers in the style and reduced numbers of ovules penetrated by pollen tubes. Reproductive isolation was weakest within the C. citriodora subsp. citriodora maternal taxon, with two hybrid backcrosses producing equivalent capsule and seed yields to the intraspecific cross. High hybridising potential was identified between all Corymbia species and F1 taxa studied. This provides opportunities for advanced generation hybrid breeding, allowing desirable traits to be amplified. It also indicates risks of gene flow between plantation and native Corymbia populations.

The influence of pre- and post-zygotic barriers on interspecific Corymbia hybridization

Corymbia species from different sections hybridize readily, with some of increasing economic importance to plantation forestry. This study explores the locations of reproductive barriers between interspecific Corymbia hybrids and investigates the reproductive success of a wide taxonomic range of C. torelliana hybrid crosses. Pollen, pistil and embryo development were investigated for four C. torelliana crosses (C. torelliana, C. citriodora subsp. citriodora, C. tessellaris and C. intermedia) using fluorescent and standard microscopy to identify the locations of interspecific reproductive isolating barriers. Corymbia torelliana was also crossed with 16 taxa, representing six of the seven Corymbia sections, both Corymbia subgenera and one species each from the related genera, Angophora and Eucalyptus. All crosses were assessed for capsule and seed yields. Interspecific C. torelliana hybridization was controlled by pre-zygotic reproductive isolating barriers inhibiting pollen adhesion to the stigma, pollen germination, pollen tube growth in the style and pollen tube penetration of the micropyle. Corymbia torelliana (subgenus Blakella, sect. Torellianae) was successfully hybridized with Corymbia species from subgenus Blakella, particularly C. citriodora subsp. citriodora, C. citriodora subsp. variegata, C. henryi (sect. Maculatae) and C. tessellaris (sect. Abbreviatae), and subgenus Corymbia, particularly C. clarksoniana and C. erythrophloia (sect. Septentrionales). Attempted intergeneric hybrids between C. torelliana and either Angophora floribunda or Eucalyptus pellita were unsuccessful. Corymbia hybrids were formed between species from different sections and subgenera, but not with species from the related genera Angophora or Eucalyptus. Reproductive isolation between the interspecific Corymbia hybrid crosses was controlled by early- and late-acting pre-zygotic isolating barriers, with reproductive success generally decreasing with increasing taxonomic distance between parent species. These findings support the monophyly of Corymbia and the close relationships of infrageneric clades. The hybridizing propensity of Corymbia species provides opportunities for breeding but suggests risks of environmental gene flow. © The Author 2012. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Comparison of three pollination methods for Eucalyptus argophloia, a small-flowered eucalypt

Context Most studies assess pollination success at capsule maturity, and studies of pre-zygotic processes are often lacking. Aims This study investigates the suitability of controlled pollination for a potential forestry plantation species, Eucalyptus argophloia, by examining pre- and post-zygotic pollination success. Methods Pollen tube development, capsule set and seed set are compared following three-stop pollination, artificially induced protogyny (AIP), AIP unpollinated and open pollination. The fecundity of stored pollen was compared with that of fresh pollen. Results Three-stop pollination, AIP and open pollination had similar numbers of pollen tubes, but AIP unpollinated had none. Open pollination produced significantly more capsules and total number of seeds than the other treatments. There were significantly more seeds per retained capsule for the open pollination and three-stop pollination treatments than for the AIP and AIP unpollinated pollination treatments. There were no significant differences relative to the age of pollen. Conclusions Pre-zygotic success in terms of pollen tubes was similar for open-pollinated, three stop and AIP, but was not reflected in post-zygotic success when the open pollination and three-stop method produced significantly more seeds per retained capsule than the AIP treatments and open pollination yielded more seeds. Capsule set and total seed set for open pollination, and fewer capsules in controlled pollinations, may reflect physical damage to buds because of the small E. argophloia flowers. Suitable alternative breeding strategies other than controlled pollinations are discussed for this species.

美味猕猴桃和软枣猕猴桃种间杂交花粉管行为和早期胚胎发生的观察

美味猕猴桃(Actinidia deliciosa(A．Chev．)C．F．Liang and A．R．Ferguson)和软枣猕猴桃（Actinidia arguta）种间传粉后，花粉管在花柱内行为的荧光观察，以及早期胚胎发生的显微观察，结果如下： 1．花粉粒在柱头的乳头细胞表面萌发，在开放型的V形花柱道内生长。 2．花粉管生长速率比对照缓慢，到达胚珠珠孔的时间平均延迟50到60小时。 3．花粉管在花柱中下部出现形态变化：部分花粉管呈波纹状弯曲;花粉管顶端膨大，尖细或破裂;花粉管直径变化;花粉管解体。 4．花粉管胼胝质沉积的变化：胼胝质沿花粉管壁不规则沉积;有的膨大的花粉管顶端出现胼胝质;有的不出现胼胝质塞，而整个花粉管壁有胼胝质分布，荧光强烈。 5．基于显微结构和种子分析，种间杂交大约有30%的胚珠能够受精，并发育成为种子，种子的胚的大小和胚乳的量与对照有差别，有约70%或更多的表现不育或败育。胚发育为茄型，受精后台子保持休眠十几天后开始横分裂。传粉后七周形成子叶胚。胚发育较对照迟缓。胚乳细胞型。 6．种间杂交能够结实，正常种子占20% -30％，败育干瘪种子占10%左右，未受精胚珠占60%- 70％。种内传粉正常种子占95%，空瘪败育种子占0.7％，未受精胚珠占3.8％。 7．种间杂交果实大小、重量，种子大小及数目，胚的大小都比对照小。

冬小麦春化作用相关基因Verc203的功能鉴定

本文应用反义基因技术对减法杂交和差异筛选所获得的春化相关基因CDNA克隆( Verc203)的功能进行鉴定。将Vcrc203插入PB1221质粒的CaMV35S启动子与GLIS基因之间构成表达载体，采用花粉管途径转基因法导入冬小麦胚，得到300粒种子。将上述处理的冬小麦种子萌发后在2-4℃处理28天，转入自然条件下栽培。以幼叶为材料提取基因组DNA，以Gus基因片断为探针，用点杂交法筛选获得转基因植株．转基因植株在自然条件下生长三个月未见抽穗， 而同样栽培条件下的对照（未经转基因处理的冬小麦，经相同条件春化处理）则全部抽穗。以Vcrc203和Gus基因的部分序列为引物，在转基因材料的基因组DNA中扩增出Vcrc203反义基因序列，Gus活性检测表明Gus基因在转基因小麦的茎尖和叶片中得到表达。上述结果表明Vcrc203的反义基因已整合到冬小麦基因组内，并得到高效表达。转基因植株的发育受到严重影响，显示Vcrc203基因可能参与了冬小麦春化作用对植物发育进程的调控．

植物克隆化基因功能分析及蛋白编码基因分离的研究

直到九十年代，以利用拟南芥菜和金鱼草突变体克隆到花器官发育基因为重要标志的植物发育研究取得了重大突破。但由于开花决定过程分子控制机理研究进展却较为缓慢。以mRNA人手利用差异筛选技术获得春化相关基因克隆(verc203，verc17)，Northem blot初步证明这些克隆对春化处理的特异性。根据反义RNA理论和技术对克隆化verc203基因功能进行研究。另外，从纯化蛋白人手，在金针菇中克隆到新的溶血性毒蛋白编码基因，并在细菌中得到表达，这一实验为利用此途径来克隆发育调控蛋白的编码基因进行了有益的技术路线探索。本研究的具体结果如下： 1． 春化相关基因(verc203)对冬小麦花发育调控 根据反义RNA的理论和技术进一步证明verc203在冬小麦开花启动和花发育过程中的可能控制作用。将verc203 DNA序列插入带有CaMV35S启动子和GUS基因的pBI221表达载体， 使之能在植物中高效表达反义RNA，并以转正义基因植株作为对照。通过花粉管途径转基因方法，得到326粒反义质粒冬小麦转化种子和1 98粒正义质粒冬小麦转化种子．所有转化处理种子和正常小麦种子萌发后，经春化处理，与未春化冬小麦植株一起培养直到对照植株达到成熟期。实验观察发现，转反义基因植株的抽穗受到明显的阻抑，开花过程大大推迟。其中表达较强的6株转基因植株甚至晚花五十多天。还发现一些表达较弱的植株尽管晚花20到30天，但花器官发育却受到影响，如，雄蕊缺失，穗发育异常等。Southemblot和PCR实验结果表明外源基因整合到转基因植株基因组。 Northrenblot以及报告基因(GUS)活性等实验证明反义基因在转反义基因植株RNA水平上得到高效表达。同时，GUS基因在蛋白质水平上得到表达。基于上述实验，有理由认为ver203基因可能是控制冬小麦春化诱导的成花启动和花发育过程中的主要基因之一。 2． 金针菇毒蛋白flammutoxin编码基因的克隆及其在细菌中的表达 利用色谱分离技术及SDS．PAGE电泳技术从金针菇(Flammuleina velutips)分离得到一种分子量为31 kDa、对人血红细胞具有溶血活性的蛋白质(flammutoxin)，通过蛋白质序列微量分析技术测得其N-端3 1个氨基酸顺序。利用氨基酸序列推测其编码基因序列信息，通过RT - PCR克隆到一个由973个核苷酸组成的编码基因，5’端93个核苷酸编码肽链序列与测得flammutoxin N-端氨基酸序列完全相同。基因序列同源性分析表明在GenBank (USA)，EMBL Database (Europe)和DDBJ (Japan)基因序列数据库中未找到与之同源的基因序列。这个基因与载体p-半乳糖苷酶部分基因编码的融合蛋白在E coli细胞中有效地得到表达。表达产物的SDS-PAGE，Western blot和溶血活性实验表明它是flammutoxin蛋白原编码基因。并对融合蛋白的溶血活性降低原因作了讨论。

花粉管通道遗传转化方法的新证据

将pBIN35S-mGFP4质粒转入受体菌DH5α中，扩繁后提取纯化质粒DNA，待棉花盛花期时，用花粉管通道转化方法将其注射到授粉24小时左右的子房中。在被检测的950个发育10～20天左右的幼胚中，发现七个幼胚在蓝光(460～490nm)激发下发出绿色荧光，而对照发出程度不同的红色荧光。将收获的“转化种子”浸泡萌发得到生长5～6天的黄化无菌苗，在200粒种子来源的无菌苗中，检出两棵转化植株。在紫外光照射灯下转化植株发出绿色荧光，其叶片和下胚轴横切面在蓝光激发下也与对照明显不同。PCR及Southern blotting结果均证实转化植株的真实性，从而为花粉管通道转化方法的可行性提供了直接可靠的细胞及分子生物学证据。 将GFPmut1质粒的gfp通过一端平接一端粘接后重组到pBI121的BamH1和Sal1限制性酶切位点从而代替GUS基因，然后将新的重组质粒用三亲交配法转入到LBA4404菌株中得到双元载体，用于棉花下胚轴切段的转化。结果表明，gfp象gus一样可作为报告基因用于农杆菌介导的棉花转化。在对筛选培养基上生长的“抗性愈伤”进一步进行报告基因检测时，只需手持紫外灯就可以检出GFP阳性愈伤，大大减少了工作量和试验费用。 另外，在进行农杆菌转化前的棉花卡那霉素敏感性实验中发现，卡那霉素对棉花下胚轴的愈伤组织形成和增殖均有明显的抑制作用，随其浓度的增加，愈伤组织形成的频率降低，增殖的倍数减小;当浓度增加到100mg/L时，愈伤组织严重褐化，其正常生长受到完全抑制;下胚轴形态学上端切段较下端更易受卡那霉素的影响。

分子调控咖啡酸-O-甲基转移酶（COMT）对木质素合成及植物生长发育的影响

本论文由两部分组成，一、构建来自小麦的COMT的反义表达载体，转化烟草，研究抑制内源COMT对植物木质素合成及其生长发育的影响;二、利用花粉管通道法，将正义和反义COMT基因转化小麦，获得转基因小麦，从而进一步分析。 一、 反义抑制COMT对植物木质素合成及其生长发育的影响 构建含有小麦的咖啡酸-O-甲基转移酶（COMT）cDNA的反义表达载体, 利用农杆菌法转化烟草。 PCR, PCR-Southern 检测显示目的基因片段成功转入烟草基因组。处于营养生长期的转基因植株表型与对照没有明显差异;而发育成熟的转基因植株的植株矮化，茎部木质素含量与对照差异不大，木质素的组成S/G比下降，部分木质部细胞发生变形。我们还发现转基因烟草种子发芽率提高，移栽2个月的子一代转基因植株光合速率、蒸腾速率有所增强。结果表明通过反义抑制COMT将影响木质素合成,并在不同的发育阶段，影响着植物的生长发育。 二、 利用花粉管通道法获得转基因小麦 将构建好的含有Bar基因的正义和反义COMT表达载体利用花粉管法转化两个小麦品种（H4564和C6001），共获得转基因处理的种子1117颗，重新播种后，发育成苗分别为321株，总成苗率为28.7%。通过除草剂PPT筛选，分别获得PPT抗性植株31株。PCR检测抗性植株，获得PCR检测阳性植株5株，总阳性率为0.45%。阳性植株分别为H4564反义处理株1株，C6001的正义和反义处理株各2株。对小麦的植株高度，分蘖数等生理性状的分析发现，转基因小麦的分蘖数减少，植株高度降低。这些生理性状的改变与COMT基因转化的关系将有待于进一步验证。